Hepatitis C virus is the leading cause of chronic liver disease throughout the world. (Boyer, N. et al. J. Hepatol. 2000 32:98-112). Patients infected with HCV are at risk of developing cirrhosis of the liver and subsequent hepatocellular carcinoma and hence HCV is the major indication for liver transplantation.
HCV has been classified as a member of the virus family Flaviviridae that includes the genera flaviviruses, pestiviruses, and hapaceiviruses which includes hepatitis C viruses (Rice, C. M., Flaviviridae: The viruses and their replication. In: Fields Virology, Editors: B. N. Fields, D. M. Knipe and P. M. Howley, Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 30, 931-959, 1996). HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.4 kb. The viral genome consists of a 5′ untranslated region (UTR), a long open reading frame encoding a polyprotein precursor of-approximately 3011 amino acids, and a short 3′ UTR. The 5′ UTR is the most highly conserved part of the HCV genome and is important for the initiation and control of polyprotein translation. The carboxyl half of nonstructural protein 5, NS5B, contains the RNA-dependent RNA polymerase.
Currently there are a limited number of approved therapies are currently available for the treatment of HCV infection. New and existing therapeutic approaches to treating HCV and inhibition of HCV NS5B polymerase have been reviewed: R. G. Gish, Sem. Liver. Dis., 1999 19:5; Di Besceglie, A. M. and Bacon, B. R., Scientific American, October: 1999 80-85; G. Lake-Bakaar, Current and Future Therapy for Chronic Hepatitis C Virus Liver Disease, Curr. Drug Targ. Infect Dis. 2003 3(3):247-253; P. Hoffmann et al., Recent patents on experimental therapy for hepatitis C virus infection (1999-2002), Exp. Opin. Ther. Patents 2003 13(11):1707-1723; M. P. Walker et al., Promising Candidates for the treatment of chronic hepatitis C, Exp. Opin. investing. Drugs 2003 12(8):1269-1280; S.-L. Tan et al., Hepatitis C Therapeutics: Current Status and Emerging Strategies, Nature Rev. Drug Discov. 2002 1:867-881; J. Z. Wu and Z. Hong, Targeting NS5B RNA-Dependent RNA Polymerase for Anti-HCV Chemotherapy, Curr. Drug Targ.—Infect. Dis. 2003 3(3):207-219.
In vitro, the polymerase activity of HCV NS5B is dependent on an RNA template and requires either a RNA or DNA primer. A variety of in vitro assays to measure the activity of the HCV NS5B polymerase have been developed. Commonly, the standard reaction mixture consists of buffers, salts, divalent cations, reducing agents, as well as nucleoside triphosphates and an RNA template and primer. The most commonly used templates and primers are synthetic homopolymeric template/primers such as poly-adenosine monophosphate:oligo-uridine monophosphate (polyA:oligo U; see, for example, S.-E. Behrens et al., EMBO J. 1996 15(l):12-22, V. Lohmann et al., J. Virol. 1997 71(11):8416-8428).
However NS5B can also initiate in vitro RNA synthesis in a primer-independent fashion when RNA templates of heteropolymeric sequence, including sequences from the HCV genome, are used. These sequences include the internal ribosome entry site located at the 5′-untranslated region of the HCV genome (HCV IRES; Kieft et al., RNA 2001 7:194-206) and the 3′-untranslated region (HCV 3′-UTR; Pellerin et al., Biochem. Biophys. Res. Comm. 2002 295:682-688). Here, the 3′-end of the template is used as the primer and elongation proceeds from a hairpin loop via a snap-back mechanism leading to a double-stranded molecule in which template and product are covalently linked.
Scintillation proximity assay (SPA) makes use of the limited pathlength of certain electron-emitters (Hart et al., Molecular Immunology 1979 16:265-267; Hart, U.S. Pat. Nos. 4,271,139 and 4,382,074; and Bertoglio-Matte, U.S. Pat. No. 4,568,649). An exemplary SPA is composed of an analyte in solution, plastic beads which scintillate when exposed to electrons, and a specific binding partner (such as an antibody) bound to the beads and specific for the analyte in solution. If the analyte incorporates a radioactive label which emits electrons of relatively short pathlength, such as tritium, the plastic beads will only scintillate when suspended in solution with the radioactive analyte when the analyte is specifically bound by the binding partner and thus localized near the surface of the beads.
SPAs have been developed and exploited for a variety of analytical purposes. SPAs have been used for radioimmunoassays, competition assays, enzyme kinetic assays, studies of ligand/receptor and antigen/antibody interactions, and studies of cellular processes (see, Cook, Drug Discovery Today 1996 1:287-294; and Cook, U.S. Pat. No. 5,665,562). The SPAs described to date all rely on specific binding interactions, such as antibody-antigen interactions, ligand-receptor interactions, biotinylated reagents which bind to streptavidin-coated beads, chelate complex formation of the species of interest, or other interactions which rely on the precise and specific structural complementarity of binding partners. While this gives SPAs high specificity for an analyte of interest, it also requires extra steps in the preparation of reagents and the time and expense of developing a binding partner system specific to the reaction of interest. It also limits its use to those systems where specific binding partners can be found or developed. For example, specific antibodies are needed for antigen-antibody assays, specific receptors are needed for ligand-receptor assays, chelate ligands must be matched to the geometry of the ion with which they form the chelation complex. If no antibodies or receptors are available for detection of a substance, specific modification of the analyte with a member of a binding pair such as biotin-streptavidin is required.
Therefore, in order to use the standard SPA assay for measuring the activity of any polymerase enzyme, including HCV NS5B polymerase, a synthetic primer such as oligo U must be modified with an affinity tag molecule (e.g. biotin), allowed to anneal to an appropriate homopolymeric template (in this case, polyA) and reacted with SPA beads coated with a molecule which can bind to the tag molecule (e.g. streptavidin). However, it would be useful and cost-effective to develop a system whereby heteropolymeric templates with either no primers or unmodified primers can be utilized in a SPA assay to measure the activity of a polymerase enzyme.